Role of Hsl7 in Morphology and Pathogenicity and Its Interaction with Other Signaling Components in the Plant Pathogen Ustilago maydis

Abstract

The phytopathogenic fungus Ustilago maydis undergoes a dimorphic transition in response to mating pheromone, host, and environmental cues. On a solid medium deficient in ammonium (SLAD [0.17% yeast nitrogen base without ammonium sulfate or amino acids, 2% dextrose, 50 M ammonium sulfate]), U. maydis produces a filamentous colony morphology, while in liquid SLAD, the cells do not form filaments. The p21-activated protein kinases (PAKs) play a substantial role in regulating the dimorphic transition in fungi. The PAK-like Ste20 homologue Smu1 is required for a normal response to pheromone, via upregulation of pheromone expression, and virulence, and its disruption affects both processes. Our experiments suggest that Smu1 also regulates cell length and the filamentous response on solid SLAD medium. Yeast two-hybrid analysis suggested an Hsl7 homologue as a potential interacting partner of Smu1, and a unique open reading frame for such an arginine methyltransferase was detected in the U. maydis genome sequence. Hsl7 regulates cell length and the filamentous response to solid SLAD in a fashion opposite to that of Smu1, but neither overexpression nor disruption of hsl7 attenuates virulence. Simultaneous disruption of hsl7 and overexpression of smu1 lead to a hyperfilamentous response on solid SLAD. Moreover, only this double mutant strain forms filaments in liquid SLAD. The double mutant strain was also significantly reduced in virulence. A similar filamentous response in both solid and liquid SLAD was observed in strains lacking another PAK-like protein kinase involved in cytokinesis and polar growth, Cla4. Our data suggest that Hsl7 may regulate cell cycle progression, while both Smu1 and Cla4 appear to be involved in the filamentous response in U. maydis.

Haplo-insufficiency for different genes differentially reduces pathogenicity and virulence in a fungal phytopathogen

“…1), indicating increased aerial hyphae associated with response to pheromone; in contrast, the smu1/Dsmu1 displayed a decrease in aerial hyphae on charcoal media. Such an increase in aerial hyphae production upon deletion of one copy of hsl7 was consistent with observations of the mating response of haploid strains bearing the deletion when mated to a wild type partner (Lovely et al, 2011; also, see Fig. S2).…”

“…Similar defects were observed for another PAK-like kinase, Cla4 (Leveleki et al, 2004). Hsl7 is a putative arginine methyltransferase that interacts with Smu1, both physically, as evidenced by yeast two-hybrid analyses, and genetically (Lovely et al, 2011). Expression of hsl7 in haploid cells has dramatic effects on cell lengths (Table S1) and evidence suggests that one of its normal roles is that of negative regulator of Smu1 (Lovely et al, 2011).…”

“…However, they fail to produce tumors through whorl infection of maize plants. In addition, we also report here that diploid cells lacking one copy of either pde1, encoding a cyclic phosphodiesterase (Agarwal et al, 2010) or hsl7, which encodes a putative arginine methyltransferase associated with cell length and cell cycle in haploid cells (Lovely et al, 2011), display altered production of aerial hyphae on charcoal agar, a trait associated with the mating response and specifically, with b-mating locus function. Such mutant diploid cells also have significantly reduced levels of virulence compared to the wild type diploid strain.…”

“…Hsl7 is a putative arginine methyltransferase that interacts with Smu1, both physically, as evidenced by yeast two-hybrid analyses, and genetically (Lovely et al, 2011). Expression of hsl7 in haploid cells has dramatic effects on cell lengths (Table S1) and evidence suggests that one of its normal roles is that of negative regulator of Smu1 (Lovely et al, 2011). Mutants of d132 diploid cells were generated in which one allele of either smu1 or hsl7 was deleted.…”

“…4) observed for the smu1/Dsmu1 mutant, the hsl7/Dhsl7 and pde1/Dpde1 mutants showed significant reduction in overall disease progression compared to both the wild type d132 and smu1/Dsmu1 mutant strains (p < 0.05, by one-way ANOVA and p < 5 Â 10 À6 , by t-test, respectively). The corresponding infections with dikaryons (where one partner had a deletion of either hsl7 or pde1) showed a trend towards reduced pathogenicity (Lovely et al, 2011;Agarwal et al, in preparation; also see Table S2, in Supplementary material). However, for hsl7, the reduction in pathogenicity was only statistically significant in the case of the haplo-insufficient mutant.…”